Mouse Serpina1a ORF clone (NM_009243)
- Name:
- pLV3-CMV-Serpina1a(mouse)-3xMyc-CopGFP-Puro
- Accession ID:
- NM_009243.4
- Antibiotic Resistance:
- Ampicillin
- Length:
- 9516 bp
- Type:
- Protein expression
- Replication origin:
- ori
- Host:
- Mammalian cells, Lentivirus
- Selection Marker:
- Puro;CopGFP
- Copy Number:
- High
- Promoter:
- CMV
- 5' Primer:
- CMV-F
- Fusion Tag:
- 3xMyc
- Growth Strain(s):
- Stbl3
- Growth Temperature:
- 37℃
- Expression Method:
- Constiutive, Stable / Transient
- Gene Synonyms:
- Aat-2; Aat2; Dom1; PI1; Spi1-1; Spi1-3
- Transcript Definition:
- Mus musculus serine (or cysteine) peptidase inhibitor, clade A, member 1A (Serpina1a), transcript variant 1, mRNA
Resources
Plasmid Protocol
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5. Store the plasmid at -20 ℃.
6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it
General Plasmid Transform Protocol
1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.
2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.
3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.
4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.
5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.
Mouse Serpina1a ORF clone (NM_009243) Sequence
LOCUS V046925 9516 bp DNA circular SYN 01-JAN-1980
DEFINITION synthetic circular DNA.
ACCESSION V046925
VERSION V046925
KEYWORDS .
SOURCE .
ORGANISM .
.
FEATURES Location/Qualifiers
LTR 234..414
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
/label="5' LTR (truncated)"
misc_feature 458..583
/note="packaging signal of human immunodeficiency virus
type 1"
/label="HIV-1 Ψ"
misc_feature 1076..1309
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
/label="RRE"
CDS 1493..1537
/codon_start=1
/note="recognized by the 2H10 single-chain llama nanobody"
/product="antigenic peptide corresponding to amino acids
655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik
et al., 2013)"
/transl_table=1
/translation="KNEQELLELDKWASL"
/label="gp41 peptide"
misc_feature 1798..1915
/note="central polypurine tract and central termination
sequence of HIV-1"
/label="cPPT/CTS"
promoter 2005..2208
/note="human cytomegalovirus (CMV) immediate early
promoter"
/label="CMV promoter"
CDS 2297..3535
/label="Serpina1a(NM_009243)"
/note="Serpina1a(NM_009243)"
/gene="Serpina1a"
CDS 3542..3631
/codon_start=1
/product="3 tandem Myc epitope tags"
/transl_table=1
/translation="EQKLISEEDLEQKLISEEDLEQKLISEEDL"
/label="3xMyc"
promoter 3675..3886
/note="core promoter for human elongation factor
EF-1α"
/label="EF-1α core promoter"
LTR 3899..4167
/note="truncated 5' long terminal repeat (LTR) from human
T-cell leukemia virus (HTLV) type 1"
/label="5' LTR (truncated)"
regulatory 4196..4205
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
/label="Kozak sequence"
CDS 4226..4888
/codon_start=1
/product="green fluorescent protein 2 from Pontellina
plumata, also known as ppluGFP2 (Shagin et al., 2004)"
/transl_table=1
/translation="PAMEIECRITGTLNGVEFELVGGGEGTPKQGRMTNKMKSTKGALT
FSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYRY
EAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDGG
YYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA"
/label="CopGFP"
CDS 4958..5011
/codon_start=1
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/product="2A peptide from Thosea asigna virus capsid
protein"
/protein_id=""
/transl_table=1
/translation="EGRGSLLTCGDVEENPGP"
/label="T2A"
CDS 5012..5611
/codon_start=1
/gene="pac from Streptomyces alboniger"
/note="confers resistance to puromycin"
/product="puromycin N-acetyltransferase"
/transl_table=1
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA*"
/label="PuroR"
misc_feature 5612..6199
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
/label="WPRE"
LTR 6273..6506
/note="self-inactivating 3' long terminal repeat (LTR)
from HIV-1"
/label="3' LTR (ΔU3)"
polyA_signal 6578..6699
/note="SV40 polyadenylation signal"
/label="SV40 poly(A) signal"
rep_origin 6718..6853
/note="SV40 origin of replication"
/label="SV40 ori"
primer_bind complement(6891..6907)
/note="common sequencing primer, one of multiple similar
variants"
/label="M13 rev"
protein_bind 6915..6931
/bound_moiety="lac repressor encoded by lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG)."
/label="lac operator"
promoter complement(6939..6969)
/note="promoter for the E. coli lac operon"
/label="lac promoter"
protein_bind 6984..7005
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
/label="CAP binding site"
rep_origin complement(7293..7881)
/direction=LEFT
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
/label="ori"
CDS complement(8052..8912)
/codon_start=1
/gene="bla"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/product="β-lactamase"
/transl_table=1
/translation="MSIQHFRVALIPFFAAFCLPVFA,HPETLVKVKDAEDQLGARVGY
IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEY
SPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDR
WEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRS
ALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGA
SLIKHW*"
/label="AmpR"
promoter complement(8913..9017)
/gene="bla"
/label="AmpR promoter"
primer_bind 9491..9507
/note="common sequencing primer, one of multiple similar
variants"
/label="M13 fwd"